By Dr. Felipe Sierra (auth.)
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Additional resources for A Laboratory Guide to In Vitro Transcription
Ifnecessary (too diluted), add 40)11 of 3 M NH4Ac and 1 ml of ethanol. 1. 2 Transcription Reaction There are essentially two variations to this protocol: the transcripts can be made radioactive for direct visualization by gel electrophoresis, or they can be made cold, and then visualized by S1 analysis or other techniques. The standard protocol uses radioactive UTP and contains O-Methyl GTP instead of GTP. This is a GTP analogue that, when incorporated into a growing RNA chain, prevents further elongation.
Add the DTT and Trasylol (but not the PMSF) to this buffer. Due to the viscosity of the solution, this requires vigorously shaking it, until no Schlieren lines are obvious. > Add 10 ml of this buffer to each SW27 tube, to form the pads. Keep these on ice at all times. > Place the Potter-Elvehjem homogenizer in the large plastic beaker with ice-water, and add a few milliliters of homogenization buffer at the bottom of the homogenizer (this is to prevent the tissue from getting stuck at the bottom).
Dialysis buffer is made fresh each time. It should be prepared well enough in advance so as to allow it to cool to 4°C before use. - Ammonium sulfate is stored either at 4 °C or at -20°C. It crystallizes, so it should be stirred for 1 or 2 hours before use (can be warmed to 37°C to re-dissolve it). This solution has occasionally been replaced by 3 M KCI, but the results are less reproducible. III Experimental 47 Step-by-Step Procedure Extract Preparation > Resuspend the nuclei in completed lysis buffer, again by bubbling and scraping with a plastic 10-ml pipet (do not use glass pipets to handle the nuclei).
A Laboratory Guide to In Vitro Transcription by Dr. Felipe Sierra (auth.)